Fusion partner for production of monoclonal rabbit antibodies

ABSTRACT

The invention provides a rabbit-derived immortal B-lymphocyte capable of fusion with a rabbit splenocyte to produce a hybrid cell that produces an antibody. The immortal B-lymphocyte does not detectably express endogenous immunoglobulin heavy chain and may contain, in certain embodiments, an altered immunoglobulin heavy chain-encoding gene. A hybridoma resulting from fusion between the subject immortal B-lymphocyte and a rabbit antibody-producing cell is provided, as is a method of using that hybridoma to produce an antibody. The subject invention finds use in a variety of different diagnostic, therapeutic and research applications.

INTRODUCTION

1. Field of the Invention

The invention relates to a fusion partner for the production of rabbitmonoclonal antibodies.

2. Background Of The Invention

It has long been recognized that rabbit antibodies have favorableproperties compared to mouse antibodies. First, rabbits are known toproduce antibodies to many antigens that are poorly immunogenic in mice(e.g., Norrby et al., Proc. Natl. Acad. Sci. 1987 84:6572-6; Raybould &Takahashi, Science 1988 240:1788-90 and Weller et al., Development 1987;100:351-63). For example, Bystryn et al (Hybridoma 1982 1:465-72)directly compared rabbit and mouse antibodies directed against humanmelanoma cells and showed that they recognize different epitopes.Second, rabbit antibodies are generally of high affinity. Third, becausemost monoclonal antibodies are generated in mice and rats, relativelyfew monoclonal antibodies are available that react with mouse or ratimmunogens. In spite of these advantages, until recently it has beenimpossible to raise monoclonal antibodies in the rabbit. Monoclonalantibodies are predominantly produced by using the hybridoma technologyoriginally developed by Koehler and Milstein (Nature 1975 256:495-7).This method involves immortalization of antibody-producing lymphocytesby fusion with myeloma cells. Since myeloma-like tumors are unknown inthe rabbit, it has been impossible to use this approach for producingrabbit monoclonal antibodies. Attempts have been made to use mousemyeloma cell lines as fusion partners (e.g., Raybould & Takahashi, 1988,supra), or to use in vitro-transformed lymphoid cell lines from rabbits,but in all cases these approaches were hampered by the instability ofthe resulting hybridomas.

A novel approach was taken by K. Knight and colleagues (Spieker-Polet etal., Proc. Natl. Acad. Sci. 1995 92:9348-52), who succeeded in producinga myeloma-like tumor in transgenic rabbits expressing oncogenes underthe control of the immunoglobulin heavy and light chain enhancers. Theyisolated a plasmacytoma cell line, termed 240E-1, from these tumors, andshowed that fusions of 240E-1 cells with rabbit lymphocytes producedhybridomas. Hybridomas were produced from lymphocytes of rabbitsimmunized using plasma proteins or whole cells, and were shown tosecrete IgG antibodies.

An improved fusion partner cell line for the production of monoclonalantibodies from rabbits is provided.

LITERATURE

References of interest include Spieker-Polet et al., Proc. Natl. Acad.Sci. 1995 92:9348-52; Rief et al., Hybridoma 1998 17:389-394; Liguori etal., 2001 Hybridoma 2001 20:189-98 and U.S. Pat. No. 5,675,063.

SUMMARY OF THE INVENTION

The invention provides a rabbit-derived immortal B-lymphocyte capable offusion with a rabbit splenocyte to produce a hybrid cell that producesan antibody. The immortal B-lymphocyte does not detectably expressendogenous immunoglobulin heavy chain and may contain, in certainembodiments, an altered immunoglobulin heavy chain-encoding gene. Ahybridoma resulting from fusion between the subject immortalB-lymphocyte and a rabbit antibody-producing cell is provided, as is amethod of using that hybridoma to produce an antibody. The subjectinvention finds use in a variety of different diagnostic, therapeuticand research applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is two panels showing that hybridoma cell lines derived from the240E-W fusion partner express variable amounts of an endogenous Ig heavychain. Hybridoma lines derived from 240E-W secrete endogenous Ig heavyand light chains. IgG proteins secreted by nine different rabbithybridoma lines were analyzed by SDS-PAGE (reducing conditions) afterpurification by protein G affinity chromatography (left-hand panel) orby western blotting with goat-anti rabbit IgG antibodies (right-handpanel). The protein bands at approx. 50 kDa correspond to Ig heavychains, the bands at approx. 25 kDa to Ig kappa light chains. Each cloneproduces a spleen-derived heavy chain (spleno-H chain) of slightlyvariable size (upper band), and a 240E-W-derived heavy chain (endo-Hchain) of constant size (lower band).

FIG. 2 shows the amino acid sequence of the variable domain of theendogenous heavy chain of 240E-W (SEQ ID NO:1), and its encoding cDNA(SEQ ID NO:2). The endo-IgH cDNA was amplified by RT-PCR from 240E-Wcell mRNA, using primers corresponding to the 5′ end of the rabbit vHsegment, combined with primers corresponding to the 3′ end of the rabbitIgG constant domain. The sequence of the mature vH domain is shown,beginning with the first amino acid following the signal peptide.

FIG. 3 is a western blot showing that endo-IgH protein was not detectedin the 240E-W2 line, while it was strongly detected in the 240E-W.240E-W2 cell lines do not express detectable endogenous Ig heavy chainprotein (endo-H chain). SDS-PAGE analysis of cell lysates prepared fromthe parent fusion partner cell line, 240E-W (lane 1), and two different240E-W2 clones (lanes 2 and 3). Ig heavy chains were detected by westernblotting with goat-anti rabbit IgG antibodies. The 240E-W2 clones arederived from a hybridoma line that was subcloned and selected for theabsence of endo-H protein (see text). Note that Ig heavy chain protein(50 kDa) is detected in the parent line 240E-W, but not in the 240E-W2clones.

FIG. 4 contains two panels of gels showing genomic PCR amplificationproducts of 240E-W2 clones, and of 240E-W cells. The results show thatthe gene encoding the endogenous IgH chain is undetectable by genomicPCR in 240E-W2 cells. The 240E-W2 cell line does not contain an intactgene encoding the endogenous Ig heavy chain (endo-IgH). Genomic PCR wasperformed using primers specific for the gene encoding the endogenous Iglight-chain (left-hand panel), or primers specific for the endo-Ig heavychain (left-hand panel). DNA was prepared from different 240E-W2subclones (lanes 1-18), or from the parent cell line, 240E-W (lanes 19).Arrows denote the expected size of the genomic fragment amplified by theprimers that were used. Results show that the endo-Ig light chain genewas equally detectable in both 240E-W and 240E-W2, whereas the endo-Igheavy chain was only detected in 240E-W (lane 19), and in DNA preparedfrom rabbit splenocytes (lane 6), but not in any of the 240E-W2 clones.Faint bands of variable sizes are likely to represent non-specificamplification products.

FIG. 5 shows a western blot demonstrating that hybridoma clones derivedfrom 240E-W2-C2 do not secrete any detectable endo-IgH. FIG. 5. Rabbithybridoma cell lines derived from the improved fusion partner cell line,240E-W2, secrete only one type of Ig heavy chain, and do not produce theendogenous heavy chain present in 240E-W cells. Supernatants from fivedifferent 240E-W2-derived hybridoma clones (lanes 1-3, 5, and 6), and asupernatant from a 240E-W-derived hybridoma (lane 4) were analyzed bySDS-PAGE followed by western blotting with goat-anti rabbit IgGantibodies. Note that the endo-IgH chain (lower band in lane 4) is notdetected in any of the 240E-W2-derived hybridoma supernatants,confirming that the endo-IgH gene has indeed been removed or inactivatedin the 240E-W2 fusion partner cell line.

DEFINITIONS

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termsare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988); Bird et al., Science, 242, 423-426 (1988);see Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986)).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or CDRs. The extent of theframework region and CDRs have been precisely defined (see, “Sequencesof Proteins of Immunological Interest,” E. Kabat et al., U.S. Departmentof Health and Human Services, (1983)). The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a rabbit monoclonalantibody may be joined to human constant segments, such as gamma 1 andgamma 3. An example of a therapeutic chimeric antibody is a hybridprotein composed of the variable or antigen-binding domain from a rabbitantibody and the constant or effector domain from a human antibody(e.g., the anti-Tac chimeric antibody made by the cells of A.T.C.C.deposit Accession No. CRL 9688), although other mammalian species may beused.

As used herein, unless otherwise indicated or clear from the context,antibody domains, regions and fragments are accorded standarddefinitions as are well known in the art. See, e.g., Abbas, A. K., etal., (1991) Cellular and Molecular Immunology, W. B. Saunders Company,Philadelphia, Pa.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like.

As used herein the term “isolated,” when used in the context of anisolated antibody, refers to an antibody of interest that is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, and even at least 99% free from other components withwhich the antibody is associated with prior to purification.

A “coding sequence” or a sequence that “encodes” a selected polypeptide,is a nucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide, for example, invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aretypically determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from viral, procaryotic oreucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA,and synthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence. Other “control elements” may also beassociated with a coding sequence. A DNA sequence encoding a polypeptidecan be optimized for expression in a selected cell by using the codonspreferred by the selected cell to represent the DNA copy of the desiredpolypeptide coding sequence.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences that areimmunologically identifiable with a polypeptide encoded by the sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given signal peptide that is operably linked to apolypeptide directs the secretion of the polypeptide from a cell. In thecase of a promoter, a promoter that is operably linked to a codingsequence will direct the expression of a coding sequence. The promoteror other control elements need not be contiguous with the codingsequence, so long as they function to direct the expression thereof. Forexample, intervening untranslated yet transcribed sequences can bepresent between the promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “nucleic acid construct” it is meant a nucleic acid sequence that hasbeen constructed to comprise one or more functional units not foundtogether in nature. Examples include circular, linear, double-stranded,extrachromosomal DNA molecules (plasmids), cosmids (plasmids containingCOS sequences from lambda phage), viral genomes comprising non-nativenucleic acid sequences, and the like.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells, which can be accomplished by genomic integration of allor a portion of the vector, or transient or inheritable maintenance ofthe vector as an extrachromosomal element. Thus, the term includescloning, and expression vehicles, as well as integrating vectors.

An “expression cassette” comprises any nucleic acid construct capable ofdirecting the expression of a gene/coding sequence of interest, which isoperably linked to a promoter of the expression cassette. Such cassettescan be constructed into a “vector,” “vector construct,” “expressionvector,” or “gene transfer vector,” in order to transfer the expressioncassette into target cells. Thus, the term includes cloning andexpression vehicles, as well as viral vectors.

The term “specific binding” refers to the ability of an antibody topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between a capture agent and analytewhen they are specifically bound in a capture agent/analyte complex ischaracterized by a K_(D) (dissociation constant) of less than 10⁻⁶ M,less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻⁹ M,less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

A polynucleotide is “derived from” a particular cell if thepolynucleotide was obtained from the cell. A polynucleotide may also be“derived from” a particular cell if the polynucleotide was obtained fromthe progeny of the cell, as long as the polynucleotide was present inthe original cell. As such, a single cell may be isolated and cultured,e.g. in vitro, to form a cell culture. A nucleotide isolated from thecell culture is “derived from” the single cell, as long as the nucleicacid was present in the isolated single cell.

A “rabbit-derived” cell is a progenitor of a cell obtained from arabbit.

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “avariable domain” includes reference to one or more variable domains andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a rabbit-derived immortal B-lymphocyte capable offusion with a rabbit splenocyte to produce a hybrid cell that producesan antibody. The immortal B-lymphocyte does not detectably expressendogenous immunoglobulin heavy chain and may contain, in certainembodiments, an altered immunoglobulin heavy chain-encoding gene. Ahybridoma resulting from fusion between the subject immortalB-lymphocyte and a rabbit antibody-producing cell is provided, as is amethod of using that hybridoma to produce an antibody. The subjectinvention finds use in a variety of different diagnostic, therapeuticand research applications.

In further describing the subject invention, the immortal B-lymphocytesof the invention will be described first, followed by a review of themethods and applications in which the subject cells find use.

IMMORTAL B-LYMPHOCYTES HAVING UNDETECTABLE IMMUNOGLOBULIN HEAVY CHAINEXPRESSION

As mentioned above, the invention provides an immortal rabbitB-lymphocyte fusion partner having undetectable endogenousimmunoglobulin heavy chain expression. The fusion partner cell may befused with an antibody-producing cell from a rabbit, e.g., an immunizedrabbit, to produce a hybridoma cell. The hybridoma cell, like the fusionpartner, produces no detectable endogenous immunoglobulin heavy chain,where “endogenous” immunoglobulin heavy chain refers to theimmunoglobulin heavy chain encoded by the parental fusion partner cell.

In certain embodiments, therefore, the subject fusion partner maycontain an altered, non-functional, immunoglobulin heavy chain gene(e.g., a heavy chain gene containing an insertion, deletion, inversionor point mutation in the coding sequence or non-coding sequence such asin the promoter or an intron of the heavy chain gene, for example; orthe heavy chain gene that may have been lost due to partial or completechromosome loss). In certain embodiments, the endogenous immunoglobulinheavy chain polypeptide has the sequence shown in FIG. 2, or a variantthereof that is at least 95%, e.g., at least 96%, at least 97%, at least98%, or at least 99% identical to that sequence and may be encoded bythe cDNA shown in FIG. 2 or a cDNA that hybridizes to and/or is at least95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99%identical to that sequence. Immunoglobulin heavy chain gene expressionmay be detected at the cDNA or protein level by RT-PCR orimmunoblotting, for example.

In certain embodiments, a subject immortal rabbit B-lymphocyte fusionpartner (or hybridoma thereof) may also exhibit undetectableimmunoglobulin light chain expression.

The fusion partner cell of the invention is further characterized inthat it is capable of fusion with a rabbit splenocyte at a highlyefficient rate (in the range of 40%-80%, or, in certain embodiments, atleast 80%), using methods essentially as described in Spieker-Polet etal. (Proc. Natl. Acad. Sci. 1995 92:9348-52), but with the modificationsdescribed in example 1. In other words, in a method that involves fusingspleen cells of an immunized rabbit with the subject fusion partner at acell ratio of 2:1 (spleen cells:fusion partner cells), plating of thosecells in a 96-well microtiter plate at approximately 2×10⁵ spleen cellsper well and selecting of HAT-resistant cells, results in approximately40%-80%, or, in certain embodiments, at least 80% or at least 90%) ofthe wells of the microtiter plate contain a hybridoma colony.

Further to the above, the subject fusion partner is also characterizedby an ability to produce hybridomas that are stable (i.e., hybridomasthat maintain the ability to produce a particular antibody for anextended time periods, e.g., at least one year). In other words, amonoclonal-antibody producing hybridoma cell line produced using asubject fusion partner can be subcloned and subcultured for manypassages, until sufficient numbers of cells are obtained to produce IgGantibody in amounts ranging from 1 milligram to 20 milligrams. In manyembodiments, the hybridoma cell line will be subcloned and subculturedto reach a cell number of 30 million, and further subcultured in ahigh-density culture system such as the Integra CellLine flask (BDBiosciences). This typically allows the recovery of five to twentymilligrams of IgG antibody from the hybridoma cell supernatant. Inparticular embodiments, amount of antibody produced after one year ofculture will be at least 70%, at least 80%, at least 90%, or, in certainembodiments at least 95% of the amount of antibody produced at thebeginning of the year.

Antibody-producing cells suitable for fusion with a subject fusionpartner include lymphocyte cells, e.g., a B lymphocyte. In a certainembodiments, spleen cells from a hyperimmunized mammal, e.g., a rabbit,are cultured with the subject fusion partner under conditions whichallow the cells to fuse. The hybridoma may be referred to arabbit-rabbit hybridoma. Such cell fusion methods are described inSpieker-Polet, supra, and, as such, are well known in the art.

Further, and depending on exactly how the subject cell is made, the cellmay further contain a recombinant construct (e.g., a retroviral vectorinserted into the genome of the cell) adapted for expression of animmortalizing (i.e., transforming) oncogene, i.e., a gene that allows acell to grow indefinitely in culture. Suitable oncogenes are well knownin the art (see, e.g., Katakura, Methods Cell Biol. 1998 57:69-91 for areview). In one exemplary embodiment, the cell may express both the mycand abl oncogenes, although a variety of other oncogenes may be readilyemployed for the same purpose. Expression of the oncogene in the cellmay by directed by an immunoglobulin gene enhancer, e.g., the Eκ or Eμ.

The subject fusion partner may be made by a variety of differentmethods. In certain embodiments, the subject fusion partner may be madeby producing a parental fusion partner using a transgenic rabbit, andthen manipulating the parental fusion partner in culture to produce afusion partner having the desired characteristics. For example, asuitable parent fusion partner may be made using known methods, e.g.,those described in Spieker-Polet, supra, and that parental fusionpartner may modified by a number of means to produce the subject fusionpartner. In one embodiment, the parental fusion partner is a cell linecalled 240E-1, as described in U.S. Pat. No. 5,675,063 andSpieker-Polet, supra, and deposited at the ATCC as accession no. HB-11870, or a cultured thereof.

In one embodiment and as described in U.S. Pat. No. 5,675,063, atransgenic rabbit is produced that contains a germ line insertion of anoncogenic transgene, and expression of the transgene results in tumorgrowth. A plasmacytoma (a tumor of B lymphocytes or plasma cells) formsas a result of the tumor growth, and the plasmacytoma may be isolatedfrom the transgenic rabbit and cultured in vitro to produce a parentalfusion partner. The plasmacytoma cells may be irradiated in the presenceof 8-azaguanine to produce and select HAT sensitive mutants.

In another embodiment, a plasma cell may be isolated from a rabbit andimmortalized in culture via introduction of a construct for expressingan oncogene. Such methods are standard in the art and reviewed inKatakura (Methods Cell Biol. 1998 57:69-91).

Once a suitable parental fusion partner is obtained, the parental fusionpartner may be modified by a number of different means to produce asubject fusion partner.

In one embodiment, the parent fusion partner may be repeatedlysub-cloned to produce the subject fusion partner. Sub-cloning methodsusually involve culturing a population of cells to produce singlecolonies, plating out cells from those single colonies onto separateplates, and re-growing the plated cells into single colonies. Thosesingle colonies can be picked, tested for a desirable property and againsub-cultured. Such methods are particularly suitable for use withunstable cell lines or cell lines that have been mutagenized. Forexample, in one embodiment, a parental fusion partner cell line can beplated out to produce single colonies, and the single colonies testedfor production of endogenous immunoglobulin heavy chain, by RT-PCR orimmunological methods, for example. In certain embodiments, up to aboutfive and in certain embodiments up to about 10 rounds of sub-culturingmay be employed.

In another embodiment, a subject fusion partner may be made from aparental fusion partner by making a targeted modification, e.g., atargeted insertion, in the immunoglobulin heavy chain locus in thegenome of the fusion partner. Since the sequence of the rabbit germlineimmunoglobulin locus is available (see, e.g., Ros et al, 2004 Gene 330,49-59) and several fragments and rearranged versions thereof have beendeposited in NCBI's PubMed database, such targeted modifications may bedone using standard methods.

Once produced, the subject fusion partner may be used as a fusionpartner in any method known in the art. For example, a subject fusionpartner may be fused to a rabbit spleen cell to produce a stablehybridoma. In one embodiment, a subject fusion partner may be fused tospleen cells of an immunized rabbit to produce a plurality ofhybridomas. Those hybridomas may be screened to select a hybridomaproducing a monoclonal antibody of interest, and cultured to produce theantibody, typically in the culture supernatant.

Procedures for immunizing animals are well known in the art as aremethods for the screening of hybrodimas and isolation of monoclonalantibodies. For example, such methods are described in Harlow et al.,(Antibodies: A Laboratory Manual, First Edition (1988) Cold SpringHarbor, N.Y.) and other references cited throughout this disclosure.

The fusion partner cell line designated below as 240E-W2 was depositedunder the terms of the Budapest Treaty with the American Type CultureCollection, 10801 University Blvd, Manassas, Va. 20110-2209, USA, onBBBBB BBBBB, 2006 and assigned ATCC Accession No. BBBBBBB.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. The examples described belowemploy the materials and methods described in Spieker-Polet (Proc. Natl.Acad. Sci. 1995 92:9348-52), which reference is incorporated byreference herein in its entirety.

Example 1

In initial experiments, 240E-1 cells were fused with spleen cellsobtained from an immunized rabbit. Following HAT medium selection,hybridoma colonies were isolated and tested for antibody production.Several antibody-secreting hybridoma clones were obtained. Afterexpansion and repeated passaging of the hybridoma cell lines, it wasfound that antibody secretion was greatly diminished in a majority ofthe clones. Similar results were reported by others (Liguori et al.,2001, supra). Furthermore, after subculturing the 240E-1 cells, theirefficiency as a fusion partner decreased. Similarly, Rief et al. (1998),supra, reported a low yield of hybridomas, and only one out of sixpositive hybridomas persisted after subcloning. The 240E-1 cell line istherefore somewhat unstable, and impractical for routine use.

240E-1 cells were subjected to repeated rounds of subcloning. Subcloneswere selected for high cloning efficiency and robust growth, as well asfor morphological characteristics, such as a bright appearance in phasecontrast microscopy and formation of uniform adherent colonies. Selectedsubclones were further tested for their ability to produce stablehybridomas. After completing four rounds of this subcloning process, astable line (termed 240E-W) was obtained. The morphological appearanceof 240E-W cells differed significantly from the parent 240E-1 cells.240E-W cells adhered more strongly to the substrate and did not formclumps. Fusion of 240E-W cells with splenocytes of immunized rabbitsconsistently yielded large numbers of stable hybridomas. In a typicalexperiment, splenocytes from a rabbit immunized with mouse tumor cellswere fused with 240E-W cells at a ratio of 2:1, following the protocoloutlined in Spieker-Polet et al., 1995, with the following modification:The cells were plated in 96-well plates, and HAT medium was added 3 daysafter plating. After two weeks, 60% of the wells contained growingcolonies. Of these, 50% produced antibodies that reacted with theimmunogen. Upon expansion and subcloning, 80% of the clones grewvigorously and retained antibody production over at least 5 passages.Selected hybridoma clones were subcultured for up to one year withoutdecrease in antibody production. Hybridoma cell lines were exceptionallyrobust and stable, and could be maintained in confluent wells forseveral days without loss of viability.

Example 2

The 240E-W cell line and the original 240E-1 cell line, express asignificant amount of an endogenous IgG heavy chain (endo-IgH), which isreadily detectable at the protein and mRNA level (FIG. 3). Theexpression of endogenous IgH persists in hybridomas derived from 240E-W,and is often equal to or higher than the expression of thesplenocyte-derived IgH (spleno-IgH) comprising the heavy chain of thedesired antibody secreted by the hybridoma (FIG. 1). As shown in FIG. 1,endo-IgH and spleno-IgH are, in most cases, readily distinguishable bySDS polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotidesequence and deduced amino acid sequence of endo-IgH was determined(FIG. 2), and the identity of the lower band in FIG. 1 as endo-IgH wasconfirmed by protein sequencing. Endo-IgH can associate with thesplenocyte-derived Ig light chain (spleno-IgL), as demonstrated by thepresence of the intact 150 kDa IgG complex on non-reducing SDS-PAGE(data not shown). Therefore, in many cases, hybridomas derived from240E-W secrete a mixture of endo-IgH/spleno-IgL andspleno-IgH/spleno-IgL. In a subset of hybridoma clones, this may resultin a loss of specificity and purity of the hybridoma-derived antibody.

It was also found that a rearranged, potentially active, gene encodingan endogenous kappa light chain (endo-IgL) is present in the 240E-W cellline, although the product of this gene could not be detected on themRNA or protein level. However, increased expression of the endo-IgLgene was observed in a subset of 240E-W derived hybridomas. Thesupernatants of these hybridoma cultures contained both endo-IgL andspleno-IgL, in variable relative amounts (see FIG. 1). The nucleotidesequence of endo-IgL was determined, and the identity of the endo-IgLband shown in FIG. 1 was confirmed by protein sequencing. Thus, it wasshown that expression of endo-IgL is undetectable in the 240E-W fusionpartner cell line, but is detectable, at variable levels, in a subset ofhybridomas derived from 240E-W. This potentially affects antibody purityand specific activity, due to the presence of mixed IgG tetramerscontaining various combinations of endo-IgH, endo-IgL, spleno IgH, andspleno-IgL.

Example 3

Fusion of 240E-W cells with rabbit splenocytes typically yields a subsetof hybridoma clones that do not secrete any detectable IgG. Since evenin the absence of secreted IgG the cells may contain intracellular IgGheavy chains, several of these clones were tested by immunocytochemistryfor the presence of intracellular IgG. Briefly, this was performed byculturing subclones in duplicate 96-well plates, and processing one ofthe plates for immunocytochemistry by drying, fixation with 0.4%paraformaldehyde, permeabilization with 0.5% Triton X-100, andimmunoperoxidase staining with polyclonal goat-anti-rabbit IgGantibodies. Several clones were identified that contained only lowlevels of IgG, and only in a subset of the cells. One of these hybridomaclones was selected for subcloning and further testing. The cells weregrown in standard growth medium ((Spieker-Polet et al., Proc. Natl.Acad. Sci. 1995 92:9348-52), and subcloned by the limiting-dilutionmethod. Single-cell subclones were expanded and again tested forintracellular IgG expression by immunocytochemistry. The subcloning wasrepeated twice, until a clone was obtained that was 100% IgG-negative.This clone was again subcloned, and it was found that all the subclonesremained 100% IgG-negative. In these subclones, IgH mRNA expression wasundetectable by RT-PCR. These clones were cultured in the presence ofazaguanine (0.13 mM) for 3 weeks to select for revertants that hadregained sensitivity to HAT medium selection. Finally, two subcloneswere selected for further analysis (240E-W2-C2 and 240E-W2-D1). Theseclones were analyzed by genomic PCR, using primers specific for theendo-IgH sequence. As shown in FIG. 4, the endo-IgH gene was notdetected in the 240E-W2 subclones, while it was strongly detected in the240E-W cell line used as a positive control. In contrast, the endo-IgLgene was equally detectable in 240E-W and 240E-W2 (FIG. 4). Thissuggests that the 240E-W2 clones have undergone a genetic deletion orrearrangement that resulted in the loss of an intact endo-IgH gene.

The 240E-W2 subclones were further characterized with regard to theirutility as a fusion partner. Fusions were performed with splenocytescollected from a rabbit immunized with a peptide derived from human p53protein, using the fusion partner cell lines 240E-W, 240E-W2-C2, or240E-W2-D1. The 240E-W2-C2 cell line gave rise to hybridomas that couldbe selected in HAT medium, and the fusion efficiency (i.e., thepercentage of wells containing hybridoma colonies) was higher than theone obtained with 240E-W (82%-100% vs. 64%). In contrast, the 240E-W2-D1cell line gave rise to hybridomas at a low efficiency (12%-22%).Further, the hybridoma wells derived from 240E-W2-C2 were shown by ELISAto contain antibodies reactive with the immunogen, at a frequencysimilar to the one observed in the 240E-W fusion. Several of thesehybridomas were subcloned and used for in vitro production of antibody,showing that hybridoma clones derived from 240E-W2 and 240E-W aresimilar in terms of stability and IgG productivity. Taken together,these results demonstrate that the 240E-W2 cell line has the propertiesexpected of an efficient fusion partner for generating rabbit monoclonalantibodies.

SDS-PAGE analysis confirmed that hybridoma clones derived from240E-W2-C2 do not secrete any detectable endo-IgH (FIG. 5). Expressionof endo-IgL was detected in only one of seven hybridoma clones derivedfrom 240E-W2-C2. The removal of endo-IgH is likely to result in improvedantibody activity and specificity.

Example 4

Comparative studies were performed to evaluate 240E-W2 with 240E-W. Both240E-W and 240E-W2 were used as fusion partners to generate hybridomas.Fusions were performed using conventional methodology. 1.5−3×10⁸lymphocytes from an immunized rabbit and the fusion partner cells weremixed at a ratio of 2:1 with PEG 4000 at (Sigma P7181) 37° C. inserum-free medium. The cells were transferred into 96-well cell cultureplates at approximately 1×10⁵ lymphocytes per well in 1640 RPMI mediumwith 15% FBS (or FCS), incubated in a CO₂ incubator. After 48 hr HATmedium (Sigma H0262) was added. Hybridoma colonies were ready forscreening in 3-5 weeks. Medium was changed 2-3 times before screening byremoving old medium and add fresh medium. Supernatants were tested forthe presence of antibody, specific for the immunogen, by ELISA. Westernblot analysis was used as a secondary screening assay. The hybridomaswere sub-cloned by limit dilution. The fusion partner 240E-W at 2×10⁴cells per well was used as feeder cells.

ELISA was performed in 96-well micro-titer plates that had been coatedovernight with immunogen at 1.0 μg/ml, plates were then saturated with2% BSA, followed by incubation with the antibody-containing supernatantfor 1 hr at room temperature.

After washing with PBS-Tween, alkaline phosphatase conjugated goatanti-rabbit immunoglobulin (IgG) was added and incubation continued foranother hour; plates were washed again and developed in the presence ofP—NPP (para-nitrophenyl phosphate). Color was read at 405 nm with aplate reader (Multiskan MCC/340 from Fisher Scientific).

Western blot analysis was performed by using whole cell lysates. Forexample, breast cancer cell line (MCF-7) lysate (50 μg/lane) was loadedon 10% SDS-polyacrylamide gel and proteins were separated byelectrophoresis. The proteins on the gel were transferred tonitrocellulose membrane. The blots were blocked with 5% skim milk inphosphate-buffered saline and incubated with the primary antibodies,followed by HRP-conjugated goat anti-rabbit and mouse IgGs (Pierce,20011). The blots were detected with ECL kit (Amersham RPN2106).

In 53 independent experiments, each for a different antibody, using240E-W2 as the fusion partner cell, the average fusion efficiencyincreased by 33%, the positive rates measured by ELISA and Western blotanalysis increased by 40% and 24% respectively; and the total successrate for identifying suitable rabbit monoclonal antibodies increases by41%, as compared with using 240E-W as the fusion partner cell.

A total of 35 hybridomas were subjected large scale production usingIntegra expression system (DB 353137). In use of these hybridomas, theaverage antibody yield increased by 32% and specific activities measuredby Western blot analysis increased by 133%, compared with hybridomasgenerated using 240E-W as fusion partner cell.

It is evident from the above results and discussion that the subjectinvention provides a important new fusion partner for the production ofrabbit monoclonal antibodies. Specifically, the subject fusion partnerallows highly efficient production of hybridoma cells that are verystable and that produce no antibodies containing heavy chains that areendogenous to the fusion partner. As such, the subject methods andsystems find use in a variety of different applications, includingresearch, therapeutic and other applications. The present inventionrepresents a significant contribution to the art.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-15. (canceled)
 16. A method of producing a monoclonal antibodycomprising: culturing a hybridoma cell produced by fusion of: a) arabbit-derived immortal B-lymphocyte, wherein immunoglobulin heavy chain(IgH) mRNA expression of said rabbit-derived immortal B-lymphocyte isnot detectable by RT-PCR and said rabbit derived immortal B-lymphocytedoes not detectably express IgH, and b) a splenocyte of a rabbit, toproduce said monoclonal antibody.
 17. The method of claim 16, furthercomprising isolating said monoclonal antibody from said hybridoma cell.18. The method of claim 16, wherein said rabbit is immunized with anantigen.
 19. The method of claim 18, wherein said monoclonal antibodyspecifically binds to said antigen.
 20. The method of claim 19, furthercomprising producing a modified version of said monoclonal antibody thatretains specific binding to said antigen.
 21. The method of claim 20,wherein said modified version of said monoclonal antibody is a humanizedantibody.
 22. The method of claim 20, wherein said modified version ofsaid monoclonal antibody retains the CDRs of said monoclonal antibody.23. The method of claim 16, comprising: culturing a plurality ofhybridoma cells produced by fusion of: a) said rabbit-derived immortalB-lymphocyte and b) a plurality of splenocytes of a rabbit, to produce aplurality of monoclonal antibodies.
 24. The method of claim 23, furthercomprising isolating said plurality of monoclonal antibodies from saidplurality of hybridoma cells.
 25. A hybridoma produced by fusing arabbit-derived immortal B-lymphocyte with a splenocyte of a rabbit,wherein immunoglobulin heavy chain (IgH) mRNA expression of saidrabbit-derived immortal B-lymphocyte is not detectable by RT-PCR andsaid rabbit derived immortal B-lymphocyte does not detectably expressIgH.
 26. The hybridoma of claim 25, wherein said rabbit is immunizedwith an antigen.
 27. The hybridoma of claim 26, wherein said hybridomaproduces a monoclonal antibody that specifically binds to said antigen.